Cell replacement therapy for damaged myocardium has been an important yet elusive goal in the treatment of heart failure. Our laboratory's overall goal is to understand the regulation of muscle cell proliferation and differentiation, and how such processes mediate myocardial development and disease. Our current approaches are to identify and characterize myocardial cells derived from human embryonic stem cells (hESCs), and to use these to both model myocardial development and explore cell therapies for myocardial diseases. Our goals for this proposal are to determine subpopulations of hESCs that develop into different cardiomyocyte types, identify the developmental stage at which hESC-derived myocardial cells most effectively engraft into host tissue, and demonstrate that hESC-derived cells can be used to improve cardiac function following myocardial injury. These relate directly to the long-range goal of PA-05-043 (Directed Stem Cell Differentiation for Cell-Based Therapies for Heart, Lung, And Blood, and Aging Diseases). The specific aims of this proposal are to: 1) identify specific subpopulations of proliferating hESCs that preferentially differentiate into cardiomyocytes; we will accomplish this by differentiating specific hESC subpopulations into cardiomyocytes in vitro, and characterizing developmental- and chamber-specific gene expression and action potential propagation; 2) determine the developmental stage at which hESC-derived myocardial cells most effectively engraft in vivo and the role of the tissue environment in cardiomyocyte subspecialization; we will accomplish this by developing reporter hESC lines and "molecular beacon" strategies for isolating myocardial cells at specific developmental time points, and determining their engraftment and differentiation in mouse myocardium in vivo; and 3) demonstrate the effects of cell therapy with hESC-derived myocardial cells in a mouse model of myocardial infarction; we will accomplish this by evaluating cardiac function and electrical conduction in a mouse model of myocardial infarction following transplant with hESC-derived myocardial cells. The proposed studies will provide new insight into human cardiac myogenesis, and offer novel approaches to myocardial regeneration. [unreadable] [unreadable] [unreadable]